Publications by authors named "Pavel Kulich"

45 Publications

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to Infection of Various Serovars of (.

Front Immunol 2021 22;12:635097. Epub 2021 Apr 22.

Department of Infectious Diseases and Preventive Medicine, Veterinary Research Institute, Brno, Czechia.

In Glässer's disease outbreaks, has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to infection with various strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of strains by post-vaccinal IgG led to enhanced phagocytosis of by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of may lead to attenuation of its virulence and pathogenicity . Together with opsonization of bacteria by these antibodies, the host may eliminate in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.
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http://dx.doi.org/10.3389/fimmu.2021.635097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101634PMC
April 2021

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Anal Bioanal Chem 2021 Jun 10;413(14):3749-3761. Epub 2021 Apr 10.

C2P s.r.o. (NEXARS), The Campus Science Park, Palachovo náměstí 726/2, 625 00, Brno, Czech Republic.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R) measured by online DLS with the R values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R/R ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.
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http://dx.doi.org/10.1007/s00216-021-03323-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035888PMC
June 2021

The first case of periorbital human dirofilariasis in the Czech Republic.

Parasitol Res 2021 Feb 8;120(2):739-742. Epub 2021 Jan 8.

Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, 621 00, Brno, Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.
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http://dx.doi.org/10.1007/s00436-020-07003-9DOI Listing
February 2021

Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies.

Pharmaceutics 2020 Dec 12;12(12). Epub 2020 Dec 12.

Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, 621 00 Brno, Czech Republic.

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.
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http://dx.doi.org/10.3390/pharmaceutics12121207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763938PMC
December 2020

A novel approach to imaging engorged ticks: Micro-CT scanning of Ixodes ricinus fed on blood enriched with gold nanoparticles.

Ticks Tick Borne Dis 2021 01 28;12(1):101559. Epub 2020 Sep 28.

Department of Pharmacology and Toxicology, Veterinary Research Institute, Hudcova 296/70, 621 00, Brno, Czech Republic.

Micro-computed tomography (micro-CT) is an exceptional imaging modality which is limited in visualizing soft biological tissues that need pre-examination contrasting steps, which can cause serious deformation to sizeable specimens like engorged ticks. The aim of this study was to develop a new technique to bypass these limitations and allow the imaging of fed ticks in their natural state. To accomplish this, adult Ixodes ricinus females were allowed to engorge in vitro on blood supplemented with PEGylated gold nanoparticles (PEG-AuNPs). In total, 73/120 females divided into 6 groups engorged on blood enriched with 0.07-2.16 mg PEG-AuNPs per ml of blood. No toxic effect was observed for any of the tested groups compared to the control group, in which 12/20 females engorged on clear blood. The ticks were scanned on a Bruker micro-CT SkyScan 1276. The mean radiodensity of the examined ticks exceeded 0 Hounsfield Units only in the case of the two groups with the highest concentration. The best contrast was observed in ticks engorged on blood with the highest tested concentration of 2.16 mg/mL PEG-AuNPs. In these ticks, the midgut and rectal sac were clearly visible. Also, the midgut lumen volume was computed from segmented image data. The reduction in midgut volume was documented during the egg development process. According to this pilot study, micro-CT of ticks engorged on blood supplemented with contrasting agents in vitro may reveal additional information regarding the engorged ticks' anatomy.
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http://dx.doi.org/10.1016/j.ttbdis.2020.101559DOI Listing
January 2021

Putative morphology of Neoehrlichia mikurensis in salivary glands of Ixodes ricinus.

Sci Rep 2020 09 29;10(1):15987. Epub 2020 Sep 29.

Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. 1946/1, 612 42, Brno, Czech Republic.

Neoehrlichia mikurensis is an emerging tick-borne intracellular pathogen causing neoehrlichiosis. Its putative morphology was described in mammalian, but not in tick cells. In this study, we aim to show the presumptive morphology of N. mikurensis in salivary glands of engorged females of Ixodes ricinus. To accomplish this, we collected I. ricinus ticks in a locality with a high N. mikurensis prevalence, allowed them to feed in the artificial in vitro feeding system, dissected salivary glands and screened them by PCR for N. mikurensis and related bacteria. Ultrathin sections of salivary glands positive for N. mikurensis but negative for other pathogens were prepared and examined by transmission electron microscopy. We observed two individual organisms strongly resembling N. mikurensis in mammalian cells as described previously. Both bacteria were of ovoid shape between 0.5-0.8 μm surrounded by the inner cytoplasmic and the rippled outer membrane separated by an irregular electron-lucent periplasmic space. Detection of N. mikurensis in salivary glands of I. ricinus suggests that this bacterium uses the "salivary pathway of transmission" to infect mammals.
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http://dx.doi.org/10.1038/s41598-020-72953-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525475PMC
September 2020

Ni and TiO nanoparticles cause adhesion and cytoskeletal changes in human osteoblasts.

Environ Sci Pollut Res Int 2021 Feb 27;28(5):6018-6029. Epub 2020 Sep 27.

Institute of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic.

Titanium-based alloys have established a crucial role in implantology. As material deteriorates overtime, nanoparticles of TiO and Ni are released. This study is focused on the impact of TiO and Ni nanoparticles with size of 100 nm on cytoskeletal and adhesive changes in human physiological and osteoarthritic osteoblasts. The impact of nanoparticles with concentration of 1.5 ng/mL on actin and tubulin expression and gene expression of FAK and ICAM-1 was studied. The cell size and actin expression of physiological osteoblasts decreased in presence of Ni nanoparticles, while TiO nanoparticles caused increase in cell size and actin expression. Both cell lines expressed more FAK as a response to TiO nanoparticles. ICAM-1 gene was overexpressed in both cell lines as a reaction to both types of nanoparticles. The presented study shows a crucial role of Ni and TiO nanoparticles in human osteoblast cytoskeletal and adhesive changes, especially connected with the osteoarthritic cells. Graphical abstract.
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http://dx.doi.org/10.1007/s11356-020-10908-8DOI Listing
February 2021

Subchronic continuous inhalation exposure to zinc oxide nanoparticles induces pulmonary cell response in mice.

J Trace Elem Med Biol 2020 Apr 6;61:126511. Epub 2020 Apr 6.

Laboratory of Neurobiology and Pathological Physiology, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Veveří 967/97, 602 00, Brno, Czech Republic; Laboratory of Neurobiology and Molecular Psychiatry, Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic. Electronic address:

Objectives: We used mice as an animal model to investigate the entry of ZnO nanoparticles from the ambient air into the lungs and other organs, subsequent changes in Zn levels and the impact on the transcription of Zn homeostasis-related genes in the lungs.

Methods: The mice were exposed to two concentrations of ZnO nanoparticles; lower (6.46 × 10 particles/cm) and higher (1.93 × 10 particles/cm), allowed to breathe the nanoparticles in the air for 12 weeks and subjected to necropsy. Characterization of the ZnO nanoparticles was done using transmission electron microscopy (TEM). Energy-dispersive X-ray (EDX) spectroscopy was used to quantify ZnO nanoparticles in the lungs, brain, liver and kidney. The total zinc content in the lungs, brain, liver, kidney, red blood cells and plasma was estimated by inductively coupled plasma mass spectroscopy (ICP-MS). Transcription rate of the genes was evaluated by RealTime PCR.

Results: The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.

Conclusion: Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.
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http://dx.doi.org/10.1016/j.jtemb.2020.126511DOI Listing
April 2020

Preparation of nanoliposomes by microfluidic mixing in herring-bone channel and the role of membrane fluidity in liposomes formation.

Sci Rep 2020 03 27;10(1):5595. Epub 2020 Mar 27.

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i., Hudcova 70, 621 00, Brno, Czech Republic.

Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.
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http://dx.doi.org/10.1038/s41598-020-62500-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101380PMC
March 2020

Gadolinium labelled nanoliposomes as the platform for MRI theranostics: in vitro safety study in liver cells and macrophages.

Sci Rep 2020 03 16;10(1):4780. Epub 2020 Mar 16.

Veterinary Research Institute, Brno, Czech Republic.

Gadolinium (Gd)-based contrast agents are extensively used for magnetic resonance imaging (MRI). Liposomes are potential nanocarrier-based biocompatible platforms for development of new generations of MRI diagnostics. Liposomes with Gd-complexes (Gd-lip) co-encapsulated with thrombolytic agents can serve both for imaging and treatment of various pathological states including stroke. In this study, we evaluated nanosafety of Gd-lip containing PE-DTPA chelating Gd prepared by lipid film hydration method. We detected no cytotoxicity of Gd-lip in human liver cells including cancer HepG2, progenitor (non-differentiated) HepaRG, and differentiated HepaRG cells. Furthermore, no potential side effects of Gd-lip were found using a complex system including general biomarkers of toxicity, such as induction of early response genes, oxidative, heat shock and endoplasmic reticulum stress, DNA damage responses, induction of xenobiotic metabolizing enzymes, and changes in sphingolipid metabolism in differentiated HepaRG. Moreover, Gd-lip did not show pro-inflammatory effects, as assessed in an assay based on activation of inflammasome NLRP3 in a model of human macrophages, and release of eicosanoids from HepaRG cells. In conclusion, this in vitro study indicates potential in vivo safety of Gd-lip with respect to hepatotoxicity and immunopathology caused by inflammation.
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http://dx.doi.org/10.1038/s41598-020-60284-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075985PMC
March 2020

Proinflammatory Effect of Carbon-Based Nanomaterials: In Vitro Study on Stimulation of Inflammasome NLRP3 via Destabilisation of Lysosomes.

Nanomaterials (Basel) 2020 Feb 27;10(3). Epub 2020 Feb 27.

Veterinary Research Institute, 62100 Brno, Czech Republic.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.
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http://dx.doi.org/10.3390/nano10030418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152843PMC
February 2020

The First Non-LRV RNA Virus in .

Viruses 2020 02 2;12(2). Epub 2020 Feb 2.

Life Science Research Centre, Faculty of Science, University of Ostrava, 71000 Ostrava, Czech Republic.

In this work, we describe the first -infecting leishbunyavirus-the first virus other than (LRV) found in trypanosomatid parasites. Its host is , a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other species.
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http://dx.doi.org/10.3390/v12020168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077295PMC
February 2020

Screening of Cellular Stress Responses Induced by Ambient Aerosol Ultrafine Particle Fraction PM0.5 in A549 Cells.

Int J Mol Sci 2019 Dec 13;20(24). Epub 2019 Dec 13.

Veterinary Research Institute, Department of Chemistry and Toxicology, Hudcova 296/70, 62100 Brno, Czech Republic.

Effects of airborne particles on the expression status of markers of cellular toxic stress and on the release of eicosanoids, linked with inflammation and oxidative damage, remain poorly characterized. Therefore, we proposed a set of various methodological approaches in order to address complexity of PM0.5-induced toxicity. For this purpose, we used a well-characterized model of A549 pulmonary epithelial cells exposed to a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Electron microscopy confirmed accumulation of PM0.5 within A549 cells, yet, autophagy was not induced. Expression profiles of various cellular stress response genes that have been previously shown to be involved in early stress responses, namely unfolded protein response, DNA damage response, and in aryl hydrocarbon receptor (AhR) and p53 signaling, were analyzed. This analysis revealed induction of GREM1, EGR1, CYP1A1, CDK1A, PUMA, NOXA and GDF15 and suppression of SOX9 in response to PM0.5 exposure. Analysis of eicosanoids showed no oxidative damage and only a weak anti-inflammatory response. In conclusion, this study helps to identify novel gene markers, GREM1, EGR1, GDF15 and SOX9, that may represent a valuable tool for routine testing of PM0.5-induced in vitro toxicity in lung epithelial cells.
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http://dx.doi.org/10.3390/ijms20246310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940800PMC
December 2019

Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders.

Pharmaceutics 2019 Dec 2;11(12). Epub 2019 Dec 2.

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i., Hudcova 70, 621 00 Brno, Czech Republic.

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.
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http://dx.doi.org/10.3390/pharmaceutics11120642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955937PMC
December 2019

Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Mol Pharm 2019 08 24;16(8):3441-3451. Epub 2019 Jun 24.

Veterinary Research Institute , Brno 62100 , Czech Republic.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00225DOI Listing
August 2019

A murine model of the effects of inhaled CuO nanoparticles on cells of innate and adaptive immunity - a kinetic study of a continuous three-month exposure.

Nanotoxicology 2019 09 23;13(7):952-963. Epub 2019 Apr 23.

Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences , Prague , Czech Republic.

The inhalation or application of nanoparticles (NPs) has serious impacts on immunological reactivity. However, the effects of NPs on the immune system are influenced by numerous factors, which cause a high variability in the results. Here, mice were exposed to a three month continuous inhalation of copper oxide (CuO) NPs, and at different time intervals (3, 14, 42 and 93 days), the composition of cell populations of innate and adaptive immunity was evaluated in the spleen by flow cytometry. The ability of spleen cells from exposed and control mice to respond to stimulation with T- or B-cell mitogens by proliferation and by production of cytokines IL-2, IL-6, IL-10, IL-17 and IFN-γ was characterized. The results showed that the inhalation of CuO NPs predominantly affects the cells of innate immunity (changes in the proportion of eosinophils, neutrophils, macrophages and antigen-presenting cells) with a minimal effect on the percentage of T and B lymphocytes. However, the proliferative and secretory activity of T cells was already significantly enhanced after 3 days from the start of inhalation, decreased on day 14 and normalized at the later time intervals. There was no correlation between the impacts of NPs on the cells of innate and adaptive immunity. The results have shown that the inhalation of CuO NPs significantly alters the composition of cell populations of innate immunity and modulates the proliferation and production of cytokines by cells of the adaptive immune system. However, the immunomodulatory effects of inhaled NPs strongly depend on the time of inhalation.
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http://dx.doi.org/10.1080/17435390.2019.1602679DOI Listing
September 2019

N-Oxy lipid-based click chemistry for orthogonal coupling of mannan onto nanoliposomes prepared by microfluidic mixing: Synthesis of lipids, characterisation of mannan-coated nanoliposomes and in vitro stimulation of dendritic cells.

Carbohydr Polym 2019 Mar 29;207:521-532. Epub 2018 Nov 29.

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i., Hudcova 70, 621 00 Brno, Czech Republic. Electronic address:

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
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http://dx.doi.org/10.1016/j.carbpol.2018.10.121DOI Listing
March 2019

Inhalation of ZnO Nanoparticles: Splice Junction Expression and Alternative Splicing in Mice.

Toxicol Sci 2019 03;168(1):190-200

*Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 14220, Czech Republic.

Despite the wide application of nanomaterials, toxicity studies of nanoparticles (NP) are often limited to in vitro cell models, and the biological impact of NP exposure in mammals has not been thoroughly investigated. Zinc oxide (ZnO) NPs are commonly used in various consumer products. To evaluate the effects of the inhalation of ZnO NP in mice, we studied splice junction expression in the lungs as a proxy to gene expression changes analysis. Female ICR mice were treated with 6.46 × 104 and 1.93 × 106 NP/cm3 for 3 days and 3 months, respectively. An analysis of differential expression and alternative splicing events in 298 targets (splice junctions) of 68 genes involved in the processes relevant to the biological effects of ZnO NP was conducted using next-generation sequencing. Three days of exposure resulted in the upregulation of IL-6 and downregulation of BID, GSR, NF-kB2, PTGS2, SLC11A2, and TXNRD1 splice junction expression; 3 months of exposure increased the expression of splice junctions in ALDH3A1, APAF1, BID, CASP3, DHCR7, GCLC, GCLM, GSR, GSS, EHHADH, FAS, HMOX-1, IFNγ, NF-kB1, NQO-1, PTGS1, PTGS2, RAD51, RIPK2, SRXN1, TRAF6, and TXNRD1. Alternative splicing of TRAF6 and TXNRD1 was induced after 3 days of exposure to 1.93 × 106 NP/cm3. In summary, we observed changes of splice junction expression in genes involved in oxidative stress, apoptosis, immune response, inflammation, and DNA repair, as well as the induction of alternative splicing in genes associated with oxidative stress and inflammation. Our data indicate the potential negative biological effects of ZnO NP inhalation.
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http://dx.doi.org/10.1093/toxsci/kfy288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390655PMC
March 2019

Pellet patented technology for fast and distinct visual detection of cholinesterase inhibitors in liquids.

J Pharm Biomed Anal 2018 Nov 25;161:206-213. Epub 2018 Aug 25.

Oritest Ltd., Nábřežní 90/4, Prague, Czech Republic.

The main objective of the presented research was to prepare an innovative carrier as a filler for detection tubes in the form of double-coated pellets with a very significant color transition during the detection of cholinesterase inhibitors such as nerve agents, organophosphorus or carbamate insecticides in liquids that is observable visually and also spectrophotometrically at 412 nm. The pellet cores were prepared by the extrusion/spheronization method. Consecutively, two different coats were applied on the pellet cores in the coating device using the Wurster column method. To increase the color change intensity, the second semipermeable coat based on Eudragit RL was applied on top of the first coat, which was formed by butyrylcholinesterase immobilized in hydroxypropyl methylcellulose. Prepared samples differing in thickness of the second coat were evaluated for their quality parameters, enzymatic activity and inhibition. The detection mechanism was based on the standard Ellman's colorimetric reaction. It was observed that the semipermeable coat prevented leaching of the enzyme into the solution and led to an increased intensity of color transition from white - yellow to white - deep yellow/orange, thus enabling a more accurate visual detection. This system allows easy, rapid and safe identification of cholinesterase inhibitors in liquids, especially chemical warfare agents.
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http://dx.doi.org/10.1016/j.jpba.2018.08.050DOI Listing
November 2018

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

Bioconjug Chem 2018 07 25;29(7):2343-2356. Epub 2018 Jun 25.

Department of Pharmacology and Immunotherapy , Veterinary Research Institute, v.v.i. , Hudcova 70 , 621 00 Brno , Czech Republic.

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00311DOI Listing
July 2018

Herpesvirus associated dermal papillomatosis in Williams' mud turtle Pelusios williamsi with effects of autogenous vaccine therapy.

J Vet Med Sci 2018 Aug 11;80(8):1248-1254. Epub 2018 Jun 11.

Veterinary Research Institute, v.v.i., Hudcova 70, 621 00 Brno, Czech Republic.

An adult female of Williams' mud turtle, Pelusios williamsi long-term captive, that was allegedly caught wild in Kenya was found to have developed papilloma-like skin lesions. Excised tumors were examined histologically after routine processing with hematoxylin and eosin (H & E) stained slides, examined for the presence of viral particles by electron microscopy employing negative staining, and examined for the presence of viral DNA by PCR. Microscopic features in pre-treatment biopsies were fully diagnostic and consistent with multifocal squamous cell papilloma. Viral-type inclusion bodies were not identified. Turtle was found to be infected by reptilian herpesvirus. Association with herpesvirus and vast multiplicity of tumors thwarted surgical solution. An autogenous vaccine was prepared using 5 g of excised fresh tissue, aseptically ground, treated with diluted formalin, centrifuged to obtain a supernatant, and subsequently exposed to UV light. Autogenous vaccine induced substantial areas of necrosis of the papillomatous lesions noted by the loss of cytological architecture, nuclear loss, and by edema. The outer edges of the healing biopsies appeared to be regenerating. Therefore, our vaccine application could be considered as effective. It is difficult to treat and eliminate herpesvirus infection because of its cryptic presence and sudden onset of disease. Successful application of autogenous vaccine could be a potentially promising strategy, which deserves further testing.
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http://dx.doi.org/10.1292/jvms.18-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115266PMC
August 2018

Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite .

Proc Natl Acad Sci U S A 2018 01 28;115(3):E506-E515. Epub 2017 Dec 28.

Life Science Research Centre, Faculty of Science, University of Ostrava, 710 00 Ostrava, Czech Republic;

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite ( and ), as well as plant-infecting was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus (LRV1/2), implying that it was acquired at about the same time the became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.
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http://dx.doi.org/10.1073/pnas.1717806115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776999PMC
January 2018

Structure and Dynamics of Alginate Gels Cross-Linked by Polyvalent Ions Probed via Solid State NMR Spectroscopy.

Biomacromolecules 2017 Aug 3;18(8):2478-2488. Epub 2017 Jul 3.

Department of Chemistry and Toxicology, Veterinary Research Institute , Hudcova 296/70, 621 00, Brno, Czech Republic.

Alginate gels are an outstanding biomaterial widely applicable in tissue engineering, medicine, and pharmacy for cell transplantation, wound healing and efficient bioactive agent delivery, respectively. This contribution provides new and comprehensive insight into the atomic-resolution structure and dynamics of polyvalent ion-cross-linked alginate gels in microbead formulations. By applying various advanced solid-state NMR (ssNMR) spectroscopy techniques, we verified the homogeneous distribution of the cross-linking ions in the alginate gels and the high degree of ion exchange. We also established that the two-component character of the alginate gels arises from the concentration fluctuations of residual water molecules that are preferentially localized along polymer chains containing abundant mannuronic acid (M) residues. These hydrated M-rich blocks tend to self-aggregate into subnanometer domains. The resulting coexistence of two types of alginate chains differing in segmental dynamics was revealed by H-C dipolar profile analysis, which indicated that the average fluctuation angles of the stiff and mobile alginate segments were about 5-9° or 30°, respectively. Next, the C CP/MAS NMR spectra indicated that the alginate polymer microstructure was strongly dependent on the type of cross-linking ion. The polymer chain regularity was determined to systematically decrease as the cross-linking ion radius decreased. Consistent with the H-H correlation spectra, regular structures were found for the gels cross-linked by relatively large alkaline earth cations (Ba, Sr, or Ca), whereas the alginate chains cross-linked by bivalent transition metal ions (Zn) and trivalent metal cations (Al) exhibited significant irregularities. Notably, however, the observed disordering of the alginate chains was exclusively attributed to the M residues, whereas the structurally well-defined gels all contained guluronic acid (G) residues. Therefore, a key role of the units in M-rich blocks as mediators promoting the self-assembly of alginate chains was experimentally confirmed. Finally, combining 2D Al 3Q/MAS NMR spectroscopy with density functional theory (DFT) calculations provided previously unreported insight into the structure of the Al cross-linking centers. Notably, even with a low residual amount of water, these cross-linking units adopt exclusively 6-fold octahedral coordination and exhibit significant motion, which considerably reduces quadrupolar coupling constants. Thus, the experimental strategy presented in this study provides a new perspective on cross-linked alginate structure and dynamics for which high-quality diffraction data at the atomic resolution level are inherently unavailable.
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http://dx.doi.org/10.1021/acs.biomac.7b00627DOI Listing
August 2017

Dynamics of mono- and dual-species biofilm formation and interactions between Staphylococcus aureus and Gram-negative bacteria.

Microb Biotechnol 2017 07 11;10(4):819-832. Epub 2017 Apr 11.

Department of Food and Feed Safety, Veterinary Research Institute, Brno, Czech Republic.

Microorganisms are not commonly found in the planktonic state but predominantly form dual- and multispecies biofilms in almost all natural environments. Bacteria in multispecies biofilms cooperate, compete or have neutral interactions according to the involved species. Here, the development of mono- and dual-species biofilms formed by Staphylococcus aureus and other foodborne pathogens such as Salmonella enterica subsp. enterica serovar Enteritidis, potentially pathogenic Raoultella planticola and non-pathogenic Escherichia coli over the course of 24, 48 and 72 h was studied. Biofilm formation was evaluated by the crystal violet assay (CV), enumeration of colony-forming units (CFU cm ) and visualization using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In general, Gram-negative bacterial species and S. aureus interacted in a competitive manner. The tested Gram-negative bacteria grew better in mixed dual-species biofilms than in their mono-species biofilms as determined using the CV assay, CFU ml enumeration, and CLSM and SEM visualization. In contrast, the growth of S. aureus biofilms was reduced when cultured in dual-species biofilms. CLSM images revealed grape-like clusters of S. aureus and monolayers of Gram-negative bacteria in both mono- and dual-species biofilms. S. aureus clusters in dual-species biofilms were significantly smaller than clusters in S. aureus mono-species biofilms.
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http://dx.doi.org/10.1111/1751-7915.12705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481519PMC
July 2017

Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices.

Front Microbiol 2016 1;7:1911. Epub 2016 Dec 1.

Veterinary Research Institute, Department of Food and Feed Safety Brno, Czechia.

The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV) are used. The present article describes the process of preparation and use of such PCV- MS2 phage-like particles (MS2 PLP) - in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific -constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables) were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV - they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.
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http://dx.doi.org/10.3389/fmicb.2016.01911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234545PMC
December 2016

Changes in the Expression of Biofilm-Associated Surface Proteins in Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants.

Biomed Res Int 2016 27;2016:4034517. Epub 2016 Oct 27.

Department of Food and Feed Safety, Veterinary Research Institute, Hudcova 70, Brno, Czech Republic.

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three biofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25-2.5% ethanol and 2500 g/mL chloramine T significantly enhanced biofilm formation. To visualize differences in biofilm compactness between biofilms in control medium, 1.25% ethanol, or 2500 g/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.
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http://dx.doi.org/10.1155/2016/4034517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102705PMC
January 2017

Prostate tumor attenuation in the nu/nu murine model due to anti-sarcosine antibodies in folate-targeted liposomes.

Sci Rep 2016 09 20;6:33379. Epub 2016 Sep 20.

Central European Institute of Technology, Brno University of Technology, Purkynova 123, CZ-612 00 Brno, Czech Republic.

Herein, we describe the preparation of liposomes with folate-targeting properties for the encapsulation of anti-sarcosine antibodies ([email protected]) and sarcosine ([email protected]). The competitive inhibitory effects of exogenously added folic acid supported the role of folate targeting in liposome internalization. We examined the effects of repeated administration on mice PC-3 xenografts. [email protected] treatment significantly increased tumor volume and weight compared to controls treated with empty liposomes. Moreover, [email protected] administration exhibited a mild antitumor effect. We also identified differences in gene expression patterns post-treatment. Furthermore, [email protected] treatment resulted in decreased amounts of tumor zinc ions and total metallothioneins. Examination of the spatial distribution across the tumor sections revealed a sarcosine-related decline of the MT1X isoform within the marginal regions but an elevation after [email protected] administration. Our exploratory results demonstrate the importance of sarcosine as an oncometabolite in PCa. Moreover, we have shown that sarcosine can be a potential target for anticancer strategies in management of PCa.
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http://dx.doi.org/10.1038/srep33379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028781PMC
September 2016

Multi-layered nanofibrous mucoadhesive films for buccal and sublingual administration of drug-delivery and vaccination nanoparticles - important step towards effective mucosal vaccines.

J Control Release 2017 03 25;249:183-195. Epub 2016 Jul 25.

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, Brno, Czech Republic. Electronic address:

Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".
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http://dx.doi.org/10.1016/j.jconrel.2016.07.036DOI Listing
March 2017

Structure and Genome Release Mechanism of the Human Cardiovirus Saffold Virus 3.

J Virol 2016 09 12;90(17):7628-39. Epub 2016 Aug 12.

Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Unlabelled: In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the uncoating of the nonenveloped human cardiovirus Saffold virus 3 (SAFV-3) of the family Picornaviridae SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 Å by X-ray crystallography and an 11-Å-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral 3- and 5-fold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many cardioviruses contain a disulfide bond formed by cysteines that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection.

Importance: A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the genome release of Human Saffold virus 3 Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus 3 particle expands, and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.
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http://dx.doi.org/10.1128/JVI.00746-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988150PMC
September 2016